Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice

Figures

• 

  Figure 1. Effects of Ech-A after treatment on MI-induced chronic heart failure in C57BL/6J mice. (A) The chemical structure of Echinochrome A, numbering of carbon atoms is represented. (B) The representative M-mode echocardiography images of LV 5 weeks in all groups. Scale bar, 0.1 s. (C–G) Quantification of 4D-mode LV ejection fraction (LVEF) (C), LV fractional shortening (LVFS) (D), LV anterior wall thickness at end diastole (LVAWd) (E), LV internal diameter at end diastole (LVIDd) (F) and end systole (LVIDs) (G) at end point (5 weeks after MI). Osmotic pump filled with Ech-A or vehicle was implanted intraperitoneally 7 days after MI. (H) The heart weight (HW)/tibia length (TL) ratio in mice at 5 weeks after MI (Sham + vehicle (●): n = 5, Sham + Ech-A 2.0 mg/kg/day (■): n = 5, MI + vehicle (○): n = 7, MI + Ech-A 0.2 mg/kg/day (▽): n = 6, MI + Ech-A 0.6 mg/kg/day (△): n = 6, MI + Ech-A 2.0 mg/kg/day (□): n = 6). p-values were calculated using one-way ANOVA followed by Šídák’s multiple comparisons test (C–H).

• 

  Figure 2. Ech-A treatment suppresses interstitial fibrosis, myocardial hypertrophy, and lipid peroxidation of LV after MI. (A) Representative whole heart section and higher magnification images stained with picrosirius red at 5 weeks after MI. Scale bar, 300 μm (upper), 100 μm (lower). (B) Quantitative result of collagen volume fractions in whole hearts and magnified remote areas. (C) Representative images of remote regions of LV tissue stained with hematoxylin and eosin at 5 weeks after MI. Scale bar, 30 μm. (D) Quantitative result of the cross-sectional area (CSA) of myocardium. (E) Representative immunofluorescence images of remote region of LV myocardium at 5 weeks after MI using anti-4-HNE antibody. Scale bar, 30 μm. (F) Semi-quantitative result of 4-HNE fluorescence intensity (F.I.). (Sham + vehicle (●): n = 5, MI + vehicle (■): n = 7, MI + Ech-A (□): n = 6). Data were presented as mean ± s.e.m. p-values were calculated using one-way ANOVA followed by Turkey’s multiple comparison test.

• 

  Figure 3. Ech-A treatment prevents RSS catabolism of LV after MI. (A,B) The representative LV section images of RSS (A) and H2S/HS−; (B) levels at 5 weeks after MI. The white dash line separates the infarct area from the non-infarct area of MI hearts. Scale bar, 300 μm. (C,D) Semi-quantitative results of fluorescent intensities in non-infarct regions of LV (A,B). (Sham + vehicle (●): n = 5, MI + vehicle (■): n = 7, MI + Ech-A (□): n = 6). Data were presented as mean ± s.e.m. p-values were calculated using one-way ANOVA followed by Turkey’s multiple comparison test.

• 

  Figure 4. Ech-A treatment concentration-dependently prevents hypoxia-induced RSS catabolism in NRCMs. (A,B) The representative NRCMs images of RSS (A) and H2S/HS− (B) levels 24 h after normoxia (21% O2) or hypoxia (1% O2) incubation with or without Ech-A (10 μM). Intracellular RSS or H2S/HS− were visualized using QS10 or SF7-AM probe, respectively. Scale bar, 30 μm. (C,D) Semi-quantitative results of QS10 FRET or SF7-AM fluorescent intensities of (A,B) (Normoxia + vehicle (●), Normoxia + Ech-A (■), Hypoxia + vehicle (○), Hypoxia + Ech-A (□), n = 5 independent experiments). (E,F) Half-maximal inhibitory concentration (IC50) of Ech-A determined by percentage of QS-10 intensity decrease (E) and SF7-AM intensity increase (F). Average probe intensity at normoxia or hypoxia condition was defined as 100% or 0% grid line, respectively. Data were presented as mean ± s.e.m. p-values were calculated using one-way ANOVA followed by Šídák’s multiple comparisons test.

• 

  Figure 5. Ech-A suppresses various stress-induced RSS catabolism in vitro. (A) Representative hiPSC-CMs images of RSS level after 24 h normoxia (21% O2) or hypoxia (1% O2) incubation with or without Ech-A concentrations (10 μM). Intracellular RSS were visualized using QS10 probe. Scale bar, 30 μm. (B) Quantitative result of QS10 FRET intensities in (A) (Normoxia + vehicle (●), Normoxia + Ech-A (■), Hypoxia + vehicle (○), Hypoxia + Ech-A (□), n = 3). (C) Representative BMDMs images of RSS level 7 h after LPS stimulation with or without different Ech-A concentrations (1 or 10 μM). Intracellular RSS were detected by SSip-1 DA probe. Scale bar, 60 μm. (D) Quantitative result of the SSip-1 DA fluorescent intensity in (C) (Control + vehicle (●), LPS + vehicle (○), LPS + 1 μM Ech-A (△), LPS + 10 μM Ech-A (□), n = 3 independent experiments). Data were presented as mean ± s.e.m. p-values were calculated using one-way ANOVA followed by Šídák’s multiple comparisons test.